Penn State University 2006:PSUprotocols
Making Cells Competent
Time: O’N then 3 hr next day
Reference: Curr. Prot. in Molec. Bio, Vol. 1, 1.8.1 "Trans. Using CaCl2"
Makes 64x50μL aliquots
- Cells (from plate)
- CaCl2 Solution
- Dry Ice (2nd floor Althouse or S. Frear)
- Prepare CaCl2 solution1
- Autoclave centrifuge bottles/tubes and chill on ice
- Before centrifuge steps, make sure centrifuge is on and at 0°C
Competent Cells: Day 0
- Streak cells on LB plate
- Grow overnight at 37°C
Competent Cells: Day 1 (optional)
- Inoculate overnight starter culture (2 mL) w/ colony from plate
Competent Cells: Day 2
- Inoculate 80 mL2 with appropriate amount of O’N to obtain OD6000~0.1. Place cells on ice at OD600=0.375 (do not allow growth past an OD of 0.4, as this decreases subsequent transformation efficiency). Typically this will take 40-60 min (check every 5 min when OD is getting near 0.375).
- Ice for 10 min.
- Transfer to a pre-chilled centrifuge tube (40mL) and spin at 1600g/7 min/0°C/no brake in small, cold rotor.
- Decant supernatant & gently resuspend pellet in 8 mL of ice-cold CaCl2 buffer.
- Spin 1100g/5 min/0°C.
- Gently resuspend pellet in 8 mL ice-cold CaCl2. Let stand on ice for 30 min.
- Spin 1100g/5 min/0°C. Decant supernatant & resuspend well in 1.6 mL ice-cold CaCl2. Cells may stand on ice for 12-24 hrs to increase competency.
- Swirl tube(s) to mix, & aliquot 50 μL into cold eppendorfs. Immerse immediately in crushed dry ice.
- Transfer to box(es) and store in -80°C freezer (downstairs on 2nd floor).
- 20 g bactotriptone
- 5 g bacto-yeast extract
- 0.5 g NaCl
- 0.19 g KCl
- Adjust to pH 7.0 w/ NaOH
- Add filter-sterilized MgCl2, MgSO4 solution to give final Mg2+ conc. of 20 mM (i.e. 10 mM MgCl2, 10 mM MgSO4)
2Procedure can be scaled up or down as necessary
Time: 20 min
- (optional) Determine plasmid concentration via A260 measurements or by comparing intensities of bands on gel with those of the ladder DNA (whose masses are known).
- To an eppendorf tube, add:
- Appropriate volume of plasmid for a total of approx. 700 ng DNA (usually, if plasmid prep is good, this will be 2 μL (i.e. ~350 ng/ μL)).
- 1 μL of appropriate 10X concentration buffer (to determine correct buffer check compatibility of restriction buffers using NEB catalogue or going online to NEB.com)
- If necessary (i.e. check NEB), add 1 μL 10X concentration BSA
- [x] μL of dH2O to make the total reaction volume in tube of 10 μL.
- Chosen restriction enzymes. They are at a high enough concentration that 0.5 μL of each is more than sufficient for a restriction digest. ALWAYS ADD THESE LAST (and work quickly), in order to minimize time out of the freezer. Keep these enzymes in their low-temp blue carrying case when out of the freezer.
- (gingerly) flick tubes, and spin down in microcentrifuge for a second
- Incubate at 37°C (in water bath or warm room) for 2-16 hrs. (3-4 hrs. optimal)
- (under review)...treat any vectors w/0.2 μL CIP and incubate for 30 min at 37°C with rest of restrictions
Time: 15 min
Reference: NEB T4 ligase technical bulletin http://www.neb.com/nebecomm/TechBulletinFiles/techbulletinM0202.pdf
- Estimate DNA concentration of restricted fragments. Aim for a 3:1 insert:vector molar ratio.
- for 20 μL reaction volume, to eppendorf add:
|10X T4 ligase buffer||2|
where x is usually 5-15 uL, y=[1,5] uL.
Also, always remember to do a negative control, which will be like the reaction above, but w/ no insert. Your transformation plates will then be an measure of any vector re-ligation/uncut background.
2. For overnight ligation, add ice to cold water in styrofoam container to bring temp to 12-14ºC. Incubate overnight (15 hr.). Alternatively, for quicker ligation, ligate at RT for 1 hr. Proceed directly to transformation.
Time: 2.25 hr
- Competent cells
- Antibiotic LB plates
- Chill ligated DNA on ice
- Thaw competent cells ((found in eppendorfs in box on second-shelf down of -80ºC freezer) on ice for ~5 min.
- Add 3 μL ligation mixture to cells in bottom of tube
- Incubate on ice for 30 min
- Heat shock in 42ºC water bath for 1 min (warm SOC media at same time).
- Incubate on ice for 2 min
- Add 500 μL warm SOC media
- Shake in 37ºC room for 1 hr
- Spread 500 μL on LB/antibiotic plates using rotator and glass spreading wand.
- Incubate plates in 37ºC room overnight.
1To make SOC, add 200 μL 500 mM filter-sterilized glucose to 5 mL SOB (20 mM final glucose concentration)